Integrated multienzyme electrochemical biosensors for monitoring malolactic fermentation in wines

Integrated amperometric biosensors for the determination of l-malic and l-lactic acids were developed by coimmobilization of the enzymes l-malate dehydrogenase (MDH) and diaphorase (DP), or l-lactate oxidase (LOX) and horseradish peroxidase (HRP), respectively, together with the redox mediator tetrathiafulvalene (TTF), on a 3-mercaptopropionic acid (MPA) self-assembled monolayer (SAM)-modified gold electrode by using a dialysis membrane. The electrochemical oxidation of TTF at +100 mV (vs. Ag/AgCl), and the reduction of TTF+ at -50 mV were used for the monitoring of the enzyme reactions involved in l-malic and l-lactic acid determinations, respectively. Experimental variables concerning the biosensors composition and the detection conditions were optimized for each biosensor. Good relative standard deviation values were obtained in both cases for the measurements carried out with the same biosensor, with no need of cleaning or pretreatment of the bioelectrodes surface, and with different biosensors constructed in the same manner. After 7 days of continuous use, the MDH/DP biosensor still exhibited 90% of the original sensitivity, while the LOX/HRP biosensor yielded a 91% of the original response after 5 days. Calibration graphs for l-malic and l-lactic were obtained with linear ranges of 5.2 × 10-7 to 2.0 × 10-5 and 4.2 × 10-7 to 2.0 × 10-5 M, respectively. The calculated detection limits were 5.2 × 10-7 and 4.2 × 10-7 M, respectively. The biosensors exhibited a high selectivity with no significant interferences. They were applied to monitor malolactic fermentation (MLF) induced by inoculation of Lactobacillus plantarum CECT 748T into a synthetic wine. Samples collected during MLF were assayed for l-malic and l-lactic acids, and the results obtained with the biosensors exhibited a very good correlation when plotted against those obtained by using commercial enzymatic kits. © 2010 Elsevier B.V. All rights reserved.The financial support of the Spanish Ministerio de Educación y Ciencia Research Project CTQ2006-02743BQUPeer Reviewe

IEEE ROC&C'2013 Centro Internacional Acapulco

The synthesis of molecular motors and their design into devices that mimic biological structures such as artificial and technological interest for many years

How to optimize the analytical performance of differential pulse voltammetry: one variable at time versus Design of Experiments

Differential pulse voltammetry has often been considered one of the most suitable techniques for electroanalytical applications. However, the voltammetric parameters used are often chosen without a proper examination of their effect on the resulting response. In this lab experiment, the students are guided to a more informed choice of the electrochemical parameters to apply depending on the application sought. In the first part of the experiment, we highlight how each voltammetric parameter affects the signal-to-noise ratio and the resolution of the voltammetric response of hydroquinone, taken as an example of the application of this electrochemical technique. A Design of Experiment is then applied to optimize the intensity and the sharpness of the oxidation peak response. Finally, an analogous approach is followed to optimize the peak resolution of an equimolar hydroquinone and catechol mixture to achieve the best separation among the peak current response for the two electrochemical processes. Thanks to these two experiments, the student will identify the correct choice of parameters to optimize as key factors for achieving the best analytical performance in specific applications

An amperometric affinity penicillin-binding protein magnetosensor for the detection of β-lactam antibiotics in milk

The preparation, characterization and performance evaluation of an amperometric affinity disposable magnetosensor, based on the use of a recombinant penicillin-binding protein (PBP) and screen-printed carbon electrodes (SPCEs), for the specific detection and quantification of β-lactam antibiotic residues in milk are reported. The PBP was immobilized onto His-Tag-Isolation-modified magnetic beads (His-Tag-Isolation-MBs), and a direct competitive assay using a tracer with horseradish peroxidase (HRP) for the enzymatic labeling was performed. The amperometric response obtained at -0.20 V vs. the Ag pseudo-reference electrode of the SPCE after the addition of H2O2 in the presence of hydroquinone (HQ) was used as the transduction signal. The developed methodology showed very low detection limits (in the low ppb level) for the 6 antibiotics tested in untreated milk samples, and a good selectivity against other antibiotic residues frequently detected in milk and dairy products. Due to the bioreceptor employed, this methodology was able to detect only the active form of β-lactam antibiotics with high affinities for both penicillins and cephalosporins. Moreover, the analysis took only 30 min. © 2013 The Royal Society of Chemistry.The financial support of the Spanish Ministerio de Economía y Competitividad Research Projects, CTQ2012-34238, and the AVANSENS Program from the Comunidad de Madrid (S2009PPQ-1642) are gratefully acknowledged. Felipe Conzuelo acknowledges a FPU fellowship from the Spanish Ministry of Education.Peer Reviewe

Redox-Polymer-Based High-Current-Density Gas-Diffusion H-2-Oxidation Bioanode Using [FeFe] Hydrogenase fromDesulfovibrio desulfuricansin a Membrane-free Biofuel Cell

The incorporation of highly active but also highly sensitive catalysts (e.g. the [FeFe] hydrogenase from Desulfovibrio desulfuricans) in biofuel cells is still one of the major challenges in sustainable energy conversion. We report the fabrication of a dual-gas diffusion electrode H-2/O(2)biofuel cell equipped with a [FeFe] hydrogenase/redox polymer-based high-current-density H-2-oxidation bioanode. The bioanodes show benchmark current densities of around 14 mA cm(-2)and the corresponding fuel cell tests exhibit a benchmark for a hydrogenase/redox polymer-based biofuel cell with outstanding power densities of 5.4 mW cm(-2)at 0.7 V cell voltage. Furthermore, the highly sensitive [FeFe] hydrogenase is protected against oxygen damage by the redox polymer and can function under 5 % O-2

Integrated amperometric affinity biosensors using Co2+-tetradentane nitrilotriacetic acid modified disposable carbon electrodes. Application to the determination of beta-lactam antibiotics

A novel strategy for the construction of disposable amperometric affinity biosensors is described in this work. The approach uses a recombinant bacterial penicillin binding protein (PBP) tagged by an N-terminal hexahistidine tail which was immobilized onto Co2+−tetradentate nitrilotriacetic acid (NTA)-modified screen-printed carbon electrodes (SPCEs). The biosensor was employed for the specific detection and quantification of β-lactam antibiotics residues in milk, which was accomplished by means of a direct competitive assay using a tracer with horseradish peroxidase (HRP) for the enzymatic labeling. The amperometric response measured at −0.20 V versus the Ag pseudoreference electrode of the SPCE upon the addition of H2O2 in the presence of hydroquinone (HQ) as redox mediator was used as the transduction signal. The developed affinity sensor allowed limits of detection to be obtained in the low part-per-billion level for the antibiotics tested in untreated milk samples. Moreover, the biosensor exhibited a good selectivity against other antibiotics residues frequently detected in milk and dairy products. The analysis time was of approximately 30 min.The financial support of the Spanish Ministerio de Economía y Competitividad Research Projects, CTQ2012-34238, and the AVANSENS Program from the Comunidad de Madrid (S2009PPQ-1642) are gratefully acknowledged. Felipe Conzuelo acknowledges an FPU fellowship from the Spanish Ministry of Education.Peer Reviewe

Affinity penicillin-binding protein amperometric biosensors for the determination of beta-lactam antibiotics in milk

Peer Reviewe